Immunosuppressive therapy and post-transplant malignancy

Organ transplantation is a well-established method for the therapy of end-stage organ failure. The emergence of novel immunosuppressive regimens has reduced the risk of rejection and extended the life expectancy of organ recipients. The long-term outcome of these patients is now challenged by life-threatening complications such as cardiovascular disease, infections and post-transplant malignancies. Malignancy is a well-recognized complication of transplantation and can manifest as de novo cancer, as a recurrence of a pre-existing malignancy or from transmission of malignancy from the donor. Recent studies show that tumour incidence increases with time after organ transplantation and is related to the intensity of immunosuppression [ 1,2 ]. Overall, a 3- to 4-fold increased incidence of cancer has been observed in transplant patients compared with age-matched controls in the general population. During immunosuppressive therapy, there is a higher frequency of some relatively rare tumours that tend to be biologically more aggressive than those that occur in the general population [ 3 ]. The relative risk for developing skin cancer in allografted patients is increased up to 70% in regions with high sun exposure [ 4 ]. Post-transplantation lymphoproliferative disorder (PTLD) occurs in up to 11% of renal transplant recipients and holds the major cause for cancer-related mortality. There is a 400- to 500-fold increase in Kaposi sarcoma compared with controls of the same ethnic origin and a 100-fold increase in vulval and anal carcinomas in transplant recipients [ 5 ]. The aetiology of post-transplant malignancy is believed to be multifactorial in nature and probably involves impaired immunosurveillance of the host, direct carcinogenic effects of some immunosuppressive agents and depressed antiviral immune activity referring to a number of common viral-related post-transplant malignancies. To minimize the risk of malignancy after transplantation, it is important to evaluate the effects of an immunosuppressive agent on cancer incidence and tumour growth. However, the common use of combination therapies limits the interpretation of single-agent effects on the tumourigenesis process. There is now growing evidence that newer immunosuppressive drugs exert anti-neoplastic effects and could therefore simultaneously address the risk of allograft rejection and cancer in transplant recipients. The focus of this review is to illustrate known pro- and anti-neoplastic mechanisms attributed to some currently used immunosuppressive drugs. The mammalian target of rapamycin (mTOR) is a critical modulator of protein translation. Emerging data indicate that mTOR plays a pivotal role in regulating a plethora of signalling pathways activated in response to growth factors and metabolic changes [ 6 ]. Inhibitors of mTOR (mTORis) exert their immunosuppressive activity by impeding the response to interleukin-2 (IL-2) and thereby blocking the activation of T- and B-cells. After binding of the mTOR inhibitor to the 12-kDa immunophilin FK506-binding protein (FKBP12), this complex interacts with and inhibits the serine/threonine kinase mTOR [ 6 ]. Currently used mTOR inhibitors in transplantation medicine include rapamycin (Rapamune®, Wyeth, Madison, NJ, USA), rapamycin-derivatives RAD001 (Everolimus®, Novartis, Basel, Switzerland) and CCI779 (Temsirolimus®, Wyeth), and the rapamycin analogue ap23573 (Deforolimus®, ARIAD Pharmaceuticals, Cambridge, MA, USA). Multiple effects of mTORis on cellular signalling have been described. Rapamycin induces inhibition of the phosphatidylinositol-3-kinase (PI3K) signalling pathway that plays a key role in regulation of proliferation, survival, mobility and angiogenesis [ 7 ]. Rapamycin further inhibits signal transducer and transcription activator 3 (STAT3) signalling [ 8 ]. STAT3 mediates the expression of a variety of genes in response to cell stimuli and is involved in many cellular processes such as cell growth and apoptosis. STAT3 is upregulated in many tumour types and elevated expression levels often correlate with a poor prognosis [ 9 ]; hence, mTOR inhibition may serve as a reasonable approach for targeting STAT3 in cancer therapy. A rare but serious complication in transplant recipients is the development of PTLD. In most cases, PTLD is associated with Epstein-Barr virus (EBV) infection of B-cells, either as a consequence of reactivation of the virus after transplantation or from primary EBV infection acquired from the donor. RAD001 was shown to inhibit the growth of Epstein-Barr virus-transformed B lymphocytes both in vitro and in vivo ; thus RAD001 therapy might prevent PTLD development [ 10 ]. In alignment with this experimental data, some patients with PTLD were treated successfully with rapamycin and the anti-CD20 antibody rituximab; however, controlled prospective randomized studies are pending [ 11 ]. Another virus-related disease, the Kaposi sarcoma, is caused by the human herpes virus 8 (HHV8). It occurs mostly in immunosuppressed patients with HIV or after solid organ transplantation. In renal transplant recipients, Kaposi sarcomas were dramatically regressed after the immunosuppressive therapy was switched from cyclosporine (CsA) to mTOR inhibitors [ 12–14 ]. One plausible molecular explanation for this phenomenon was recently provided by Sodhi et al . They showed that HHV8 encodes a G protein-coupled receptor (vGPCR) that activates the mTOR pathway via phosphorylation of tuberin (TSC2). The inhibition of mTOR with rapamycin prevented vGPCR-induced sarcomagenesis, thus supporting the proposed promoting role of the activated mTOR pathway in endothelial cell transformation [ 15 ]. Further, mTOR inhibitors were shown to exert potent antiangiogenic activity in vitro and in vivo in primary and metastatic tumours via inhibition of VEGF production [ 16 ]. This antiangiogenic activity of mTORis might at least in part contribute to the regression of the highly vascularized Kaposi sarcomas. Evidence for anti-neoplastic effects of mTOR inhibitors are further provided by recent clinical cancer trials. The most encouraging data were reported for mTOR administration in mantle cell lymphoma [ 17 ] and renal cell carcinoma [ 18,19 ]. Additional ongoing cancer trials include malignant melanoma, hepatocellular carcinoma, glioblastoma, lung cancer, breast cancer and other tumour entities [ 20 ]. In contrast to their status in clinical oncology, in the setting of organ transplantation, mTORis are anticipated to exert a dual role by providing adequate immunosuppression to prevent organ rejection and simultaneously impair tumour development. A multivariate analysis of renal transplant patients examined the incidence of post-transplant malignancies in patients receiving mTOR inhibitors alone (everolimus or sirolimus), calcineurin inhibitors alone (CsA or tacrolimus) or a combination of both types of agents. Maintenance of immunosuppression with the mTOR inhibitor drugs sirolimus and everolimus alone or even in combination with calcineurin inhibitors (CNI) showed a significantly reduced risk of developing any post-transplant de novo malignancy and non-skin solid malignancy compared to a therapy with CNI alone [ 21 ]. Analysis of five multicentre studies of patients after renal transplantation revealed that patients receiving a rapamycin-based therapy without CsA or a rapamycin maintenance therapy after early CsA withdrawal had a significantly lower rate of malignancy in the first 2 years after transplantation [ 22 ]. Recent clinical trials in renal transplant recipients have further confirmed the significantly reduced risk of developing any malignancy under treatment with mTOR inhibitors [ 23,24 ]. However, in the Symphony study that investigated the efficacy and relative toxicity of four different immunosuppressive regimens in 1645 renal transplant recipients, the highest cancer incidence was noted in the rapamycin group after 12 months of follow-up [ 25 ]. Azathioprine (AZA) is a purine analogue that is incorporated into cellular DNA where it inhibits the purine nucleotide synthesis and interferes with RNA synthesis and metabolism. Its strong correlation with tumour incidence in transplant recipients is reflected by the fact that it is listed as a human carcinogen in the 11th Report on Carcinogens of the US Department of Health and Human Services [ 26 ]. Two large prospective epidemiological studies reported a high incidence of non-Hodgkin's lymphoma, squamous cell cancers of the skin, hepatobiliary carcinomas and mesenchymal tumours in renal transplant patients, who were treated with AZA and prednisone [ 26 ]. The high incidence of skin cancers in the course of treatment with AZA is proposed to be due to its synergism with UV radiation in carcinogenesis. It has been shown that AZA causes the accumulation of 6-thioguanine in patients’ DNA, and 6-thioguanine and UVA are synergistically mutagenic [ 27 ]. Furthermore, the intercalation of thioguanine into the DNA inhibits repair and induces codon misreads that could encounter for AZA-mediated cytotoxicity and carcinogenesis [ 28 ]. However, in the era of combination therapies the effects of lower doses of AZA on tumour development in a transplanted patient are difficult to determine. Compared with the more powerful immunosuppressant CsA, AZA has been associated with a lower cumulative incidence of tumours after transplantation [ 29 ]. Hence, a clear correlation of less intensive AZA use with cancer development in transplant recipients has not been established. Mycophenolic acid (MPA) reversibly inhibits inosine 5′-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. MPA displays high lymphocyte specificity and cytotoxicity due to the higher dependence of activated lymphocytes on both salvage and de novo synthesis of guanine nucleotides relative to other cell types. The morpholinoethyl ester of MPA, mycophenolate mofetil (MMF), has potent immunosuppressive properties and is used worldwide in transplantation medicine to prevent organ rejection. In addition to the well-known immunosuppressive effects, an increasing number of studies document the antiproliferative properties of MPA on a variety of cell lines [ 30 ]. IMPDH activity is increased significantly in cancer cells, and it was thus considered a sensitive target for chemotherapy [ 31 ]. In fact, MPA has been shown to inhibit the growth of tumour cells in vitro and in mouse xenografts [ 32 ]. This observation led to testing of MPA in small cohorts of patients with a variety of cancers in the 1970s. Considerable dose-limiting gastrointestinal toxicity limited the success of these trials due to the use of the diethanolamine salt of MPA rather than the currently prescribed MMF prodrug [ 33,34 ]. One molecular mechanism contributing to MPA's antitumour activity might be its ability to induce apoptosis as demonstrated in human neuroblastoma-, B-lymphoma- and multiple myeloma cells [ 35–37 ]. Furthermore, the antitumour activity of MPA is linked to its antiangiogenic properties. MPA was found to potently inhibit endothelial cell proliferation in vitro and block tumour-induced angiogenesis in vivo [ 38 ]. We have recently shown a preferential sensitivity of endothelial cells to MPA treatment compared to different tumour cell lines. A glioblastoma cell line (U87) displayed resistance to MPA treatment in vitro but MPA significantly inhibited U87 tumour growth in vivo suggesting an important role for the tumour microenvironment in MPA response [ 39 ]. In another study that used RNA interference, the knockdown of one of the two known isoforms of inosine monophosphate dehydrogenase (IMPDH-1) was sufficient to cause endothelial cell cycle arrest. In addition to that data, anti-endothelial activity of MPA was detected in two recent pharmacological screens among FDA-approved drugs that aimed to identify compounds with antiangiogenic properties [ 40,41 ]. The interaction of MMF with adhesion molecules seems to play an important role in preventing integrin-dependent tumour dissemination and metastasis. MMF was shown to induce alterations of the beta1 integrin profile and thereby block tumour cell adhesion to vascular endothelium [ 42 ]. Another mechanism discussed for the antitumour activity of MPA is the induction of differentiation in tumour cells. Addition of MPA to cultures of HL-60, neuroblastoma and melanoma cells resulted in a decreased cellular level of guanine nucleotides and the induction of cell differentiation [ 43–45 ]. In a phase I clinical trial, MPA was tested as an anticancer agent in patients with advanced multiple myeloma. Partial response to the drug adds evidence that inhibition of the IMPDH pathway may be a promising novel targeted therapy to overcome drug resistance in multiple myeloma [ 46 ]. Furthermore, synergy between imatinib and MPA in the treatment of chronic myleogenous leukaemia (CML) has been reported [ 47 ]. Evidence of potential anti-neoplastic effects of MPA in transplant recipients are provided by a number of clinical studies. In a large cohort study that examined the risk of malignancies with MMF in renal transplant recipients, a trend towards a lower risk of malignancy and a significant increase in time to malignancy in the MMF group were found. [ 48 ]. Data from the transplant registry of the International Society for Heart and Lung Transplantation (ISHLT) showed in a multivariate analysis that the use of MMF in standard immunosuppressive regimens is associated with a significantly lower risk of developing malignancy after orthotopic heart transplantation [ 49 ]. An independent report compared the cancer incidence in AZA- versus MMF-treated patients using data from the SRTR database [the SRTR database contains information collected by the Organ Procurement and Transplantation Network (OPTN)]. In 17 145 adult renal transplant recipients with pre-existing diabetes mellitus, the incidence of any post-transplant malignancy was higher in AZA-treated patients compared with MMF-treated patients (3.7% versus 2.2%, respectively). This report also indicated a significant difference in lymphoproliferative malignancies (0.6% versus 0.3%) and de novo solid tumours (2.5% versus 1.6%) [ 50 ]. However, the Tricontinental Multicenter study and the US Randomized study that compared the treatment of MMF with AZA in cadaveric renal recipients showed no difference in the incidence of any malignancy between the two drugs [ 51,52 ]. Since MMF is more effective than AZA in preventing allograft rejection [ 53 ] and might even lower the risk of malignancy, it offers a favourable profile in transplantation medicine. The immunosuppressive agent FTY720 is a synthetic analogue of sphingosine and was isolated from culture filtrates of the ascomycete Isaria sinclairii . FTY720 inhibits the egression of lymphocytes from lymph nodes to efferent lymphatics and blood via modulation of sphingosine-1-phosphate (S1P) receptors on lymphocytes [ 54 ]. The efficacy and safety of FTY720 in de novo kidney transplant patients was proven in combination with CsA and corticoids in a phase II study [ 55 ]. However, the multifaceted mode of action of FTY720 opens up other possibilities for its use, e.g., as anti-cancer agent. FTY720 was shown to exert potent anti-tumour and anti-metastatic activities in different tumour types in vitro and in vivo. Pro-apoptotic activity of FTY720 was linked to its ability to inhibit Akt- or downregulate Bcl2 survival signalling [ 56 ]. In multiple myeloma cell lines, Yasui and colleagues showed that FTY720 induces mitochondria-mediated apoptosis followed by the release of mitochondria proteins, cleavage of PARP and activation of caspase-8, -3 and -9. FTY720 also inhibits other survival signals triggered by cytokines such as IL-6 via inhibition of STAT3, Akt and ERK phosphorylation in a dose-dependent manner [ 57 ]. The potent antiangiogenic activity of FTY720 is linked to its functional antagonism of vascular S1P receptors and transactivation of the CXCR4 chemokine receptor [ 58,59 ]. Recent studies showed that FTY720 decreases vascular permeability induced by the key angiogenic protein VEGF [ 60 ]. Azuma and colleagues showed that FTY720 caused cytoskeletal changes in cancer cells that resulted in mis-shaped cells with reduced filopodias. In addition, it reduced the expression of integrins on the surface of cancer cells and impeded their ability to adhere and migrate to extracellular matrix proteins [ 61 ]. Evidence that FTY720 could inhibit cancer metastasis process comes from a study of Zhou et al . in 2006. They showed that FTY720 markedly inhibited prostate cancer cell invasion by down-regulating GTP-bound RhoA, a protein that is associated with metastasis in many types of malignancies [ 62 ]. Recent published data support the investigation of FTY720 as a novel therapeutic approach for patients with blast crisis in chronic myelogenous leukaemia (CML) or adult acute lymphoblastic leukaemia that express the BCR-ABL oncogene. The prognosis in these patients is usually poor. FTY720 was shown to induce cell death and reduce clonogenicity in myeloid and lymphoid cell lines and patient-derived progenitor cells that were either sensitive or resistant to imatinib and/or dasatinib. Of note, no adverse effects of FTY720 on normal bone marrow progenitor cells were detected in this study [ 63 ]. Although Novartis discontinued the development of FTY720 for allograft recipients due to a lack of benefits compared with MMF in a large phase III clinical trial, the multifaceted activities of this compound described here warrant further clinical testing. CsA and tacrolimus are among the most widely used immunosuppressive agents belonging to the family of CNI. Their major effect is to block the expression and to increase the production of growth thereby immune response [ ]. are linked to a higher incidence and aggressive of effects were attributed to the of the organ immune [ ]. However, and colleagues were to that cells significant induction of increased cell and thereby cancer by a mechanism probably by [ ]. is a that has a dual role in carcinogenesis. it as a tumour and causes growth of cells and cells in the early of cancer [ ]. In an effect that is favourable for the survival, and metastasis of the tumour [ ]. data that effects of CsA might rather the growth of tumours than de novo mechanisms for de novo cancer development induced by CsA include its ability to inhibit the DNA repair that the accumulation of DNA [ ]. with of activated this could in decreased of cells. The of clinical studies the risk of cancer development in patients treated with are In a randomized patients receiving CsA had an increased incidence of post-transplant malignancy, as compared with those receiving doses versus of patients, respectively). This trend was for types of skin cancer, PTLD and other cancers [ ]. In a of patients with hepatocellular carcinoma who transplantation, a higher cumulative of CsA showed a effect on the survival rate [ ]. The of cancer incidence in patients treated with CsA versus tacrolimus Although tacrolimus is associated with a higher immunosuppressive [ there are studies that report a in the incidence of de novo solid tumours these patients were treated with tacrolimus versus CsA [ ]. the other tacrolimus treatment was associated with a higher rate of PTLD that might be with its immunosuppressive The analysis of renal allograft recipients in the of demonstrated in patients who not induction therapy, the cumulative incidence of PTLD was lower in patients than in This difference the patient had induction therapy, the recipients to a higher risk for PTLD development. A explanation could be the strong immunosuppressive effect of induction therapy that might the among the maintenance drugs [ ]. are associated with a higher risk of post-transplant malignancy, in with induced Furthermore, the use of induction regimens is one of the risk factors for the development of PTLD. A in the frequency of PTLD was first described in in heart transplant recipients, who the antibody in addition to standard immunosuppressive therapy [ ]. studies the effect of and on PTLD development this data [ ]. In treatment with receptor such as or was not with an increased risk for post-transplant malignancy [ 2 ]. target activated and the number of lymphocytes the a recent study has the of different induction therapies on survival and the incidence of lymphoma in renal allograft recipients. The highest of lymphoma were found in patients treated with or receptor the with to survival and lymphoma incidence [ ]. 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